Wide confocal cytometry: a new approach to study proteomic and structural changes in the cell nucleus during the cell cycle
Wide confocal cytometry: a new approach to study proteomic and structural changes in the cell nucleus during the cell cycle
复制标题
DOI：
10.1007/s00418-007-0352-0
复制
发表时间：
2008-01-01
影响因子：
2.3
通讯作者：
Buckle, Veronica
中科院分区：
文献类型：
--
作者：
Iborra, Francisco J.;Buckle, Veronica
宽场共聚焦细胞术（WCC）是本文所开发的一种新方法，它利用标准共聚焦系统收集细胞内容物及精细结构细节的定量信息。该系统在非共聚焦条件下运行，以便捕获样本发出的最大量的光（与激光扫描细胞术相当）。在对大分子含量（DNA、RNA、特定蛋白质、脂质等）进行分析之后，可以使用常规共聚焦显微镜对细胞进行取样。我们分析了WCC的照明和采集能力。通过对海拉细胞的细胞周期阶段进行分析，观察DNA含量以及S期和有丝分裂的标志物，验证了WCC的定量能力。作为这种方法潜在应用的一个例子，我们记录了细胞周期中细胞核的变化。有丝分裂后，细胞核的形状从细长变为椭圆形，并一直保持到G2期。这种变化与核体积增加有关。随着核体积的增加，染色质以等比例的方式解聚，这可能是由于基因表达的增加以及RNA代谢所必需的因子。
Wide-confocal-cytometry (WCC) is a new method developed in this paper that uses a standard confocal system to gather quantitative information on contents and fine structural details of cells. The system is operated under conditions of non-confocality, in order to capture the maximum amount of light emitted by the specimen (comparable to LSC). After analysis of macromolecule content (DNA, RNA, specific proteins, lipids, etc.), cells can be sampled using conventional confocal microscopy. We analyzed the illumination and acquiring capabilities of WCC. The quantitative power of WCC was validated by analysis of cell cycle stage in Hela cells, looking at DNA content and markers for S phase and mitosis. As an example of the potential of this methodology we have documented changes in cell nucleus during the cell cycle. After mitosis the cell nucleus changes its shape from elongated to ellipsoid and remains constant until G2. This change is associated with nuclear volume increase. As nuclear volume increases, chromatin becomes decondensed in an isometric manner, probably due to the increase in gene expression and factors necessary for RNA metabolism.